Hydrophobins are small, secreted cysteine-rich amphipathic proteins found in fungi. Mature hydrophobin proteins, with secretion signal peptides removed, are generally small proteins that are around 100 amino acids in length. Hydrophobins are characterized by having a pattern of 8 highly conserved cysteines that are positioned within the mature protein in a 1-2-1-1-2-1 pattern (dashes represent variable numbers of amino acids). Pairs of these 8 cysteines form disulfide bonds, resulting in 4 disulfide bonds that are necessary for the proper folding of the protein. Though the 8 cysteines are highly conserved, different hydrophobins are quite variable in their amino acid sequences, some having as little as 11 % or less sequence identity (Wessels, J. G. H. 1994 Annu. Rev. Phytopathol. 32:413-37). Currently, over 50 hydrophobin sequences from a range of fungal species are known, and are available, for example, through GenBank and the National Center for Biotechnology Information (NCBI; Bethesda, Md.). Many fungal species have several reported hydrophobins. Based on differences in protein primary structure, hydrophobins can be grouped into two distinct groups, Class I and Class II.
Genetic studies have implicated hydrophobins as playing roles in providing fungal surface properties. Some hydrophobins, belonging to Class I, are known to form rodlet protein layers found on fungal surfaces. Rodlet layers are extremely hydrophobic and are responsible for generating fungal surface hydrophobicity. Thus all fungal surfaces including aerial hyphae, fruiting bodies and vegetative spores, e.g., conidia, are coated with the hydrophobic domain of self-assembled hydrophobins exposed to the environment and the hydrophilic domain attached or interacting with the hydrophilic cell wall. Thus some roles of hydrophobins in nature are to allow fungal hyphae to escape into the air from its aqueous environment and search for new food sources and surfaces, to line the internal air spaces in Schizophyllum commune fruiting bodies in order to prevent flooding by water of these air channels, and to promote interactions between the fungus and the plant during pathogenicity, either acting as elicitors of plant defense responses or as stealth factors protecting the invading fungus from detection and rejection by the plant (Whiteford, J. R., P. D. Spanu 2002 Molec. Plant Pathol. 3: 391-400).
The best characterized Class I hydrophobin, SC3, which is obtained from the fungus Schizophyllum commune, has been used to study hydrophobin assembly on surfaces and the resulting changes in surface properties. Two Class II hydrophobins, HFBI and HFBII, isolated from Trichoderma reesei, have also been studied. These hydrophobins have been found to be useful as surface coatings to alter the properties of the coated surfaces. The amphipathic properties of hydrophobins, meaning having both hydrophobic and hydrophilic properties, allow them to interact with a wide range of material surfaces. In general, upon contact with either hydrophobic or hydrophilic surfaces, hydrophobin monomers self-assemble to form a film covering the surface. A consequence of hydrophobin film formation is a change in surface wettability. Thus on hydrophobic surfaces such as Teflon® film, a hydrophobin coating can increase hydrophilicity as measured by a decrease in the water contact angle. Conversely, on hydrophilic surfaces hydrophobin coatings can decrease wettability, measured by an increase in the water contact angle. Particularly Class I hydrophobin SC3 from the fungus Schizophyllum commune and Class II hydrophobins HFBI and HFBII from Trichoderma reesei have been used in coating studies. HFBI and HFBII formed weakly ordered and highly crystalline coatings, respectively, on water surfaces (Sermaa, R. et al. Appl. Crystallography 36:499-502). Properties of different hydrophobins, even among hydrophobins within the same Class, can be quite variable in different coating situations.
The novel properties of hydrophobins, including the ability to self-assemble at interfaces in aqueous solution and under mild conditions, immediately suggest potential applications for these surface active proteins. Some potential applications presented in the literature include:                use in tissue engineering, particularly in coating of unnatural surfaces with a natural protein to increase the unnatural surface biocompatability, e.g., medical implants and surgical instruments (Wessels, J. G. H. 1997 Adv. Microbiol. Physiol. 38: 1-45).        use in drug delivery, especially the delivery of hydrophilic drugs. Drug oil vesicles coated with a hydrophobin would permit attachment of targeting antibodies to the outside of these vesicles (Wessels, J. G. H. 1997 Adv. Microbiol. Physiol. 38: 1-45).        use in the formation of stable foams in food manufacturing and as a natural surface-active agent in hair products (Wessels, J. G. H. 1997 Adv. Microbiol. Physiol. 38: 1-45, Kershaw, M. J., J. Talbot. 1998 Fung. Genet. Biol. 29: 18-33).        use to pattern different molecules on a surface with nanometer accuracy (Scholtmeijer, K., et al. 2001 Appl. Microbiol. Biotechnol. 56: 1-8).        use to bind or immobilize factors on a hydrophobic support, such as yeast cells expressing a hydrophobin in its cell wall (Nakari-Setala, T., et al. 2002 Appl. Environ. Microbiol. 68: 3385-3391), and several commercially available lipases (Palomo, J. M., et al. 2003 Biomacromol. 4:204-210).        use to develop novel diagnostic sensors through hydrophobin self-assembly on electrode surfaces (Bilewicz, R., et al. 2002 J. Phys. Chem. B. 105: 9772-9777).        
Hydrophobins used in previous studies have generally been isolated from the natural fungus in which the hydrophobin was identified. However, the amounts of protein that can be obtained using this method of preparation are inadequate for commercial applications. A successful recombinant DNA technology method would be required to supply adequate amounts of hydrophobin proteins for commercial uses. An example of recombinant hydrophobin expression is in WO 00/058342, where the endogenous HFBI hydrophobin was overexpressed in Trichoderma reesei. Using a recombinant DNA technology method also allows the creation of hydrophobin variants that may provide superior properties for use in specific applications.
Methods of treating surfaces with hydrophobins to provide a stable coating have been described. In US20030134042, Teflon® materials were incubated in an SC3 hydrophobin solution at 25° C. and then a heat treatment was applied. A heat treatment of 63° C. or 70° C. in the presence of different detergents at a concentration of 0.1% was required to obtain strong binding of the hydrophobin to the Teflon® surface. Heat treatments at lower temperatures, but above 30° C., in the presence of detergent were partially effective in transitioning the SC3 hydrdphobin to a beta-sheet state deemed necessary for strong binding to hydrophobic surfaces.
Another method of treating surfaces requires pretreatment of the hydrophobin prior to application. In US200301 13454 the disulfide bonds of the SC3 hydrophobin were disrupted by treating with sulphite to add sulfite groups, or with reducing agent followed by a sulfhydryl-protecting agent, or with a reducing agent followed by maintenance in a non-oxidizing environment. The thusly treated hydrophobin was then used to coat Teflon® materials. In a third step the hydrophobin coated material was treated to remove sulfhydryl-protecting groups if present, and with an oxidizing agent to reform disulfide bridges.
There is thus a need for identification of hydrophobins that are useful in the types of applications described above, as well as in additional applications. Also there is a need for the isolation of genes encoding useful hydrophobins such that recombinant DNA technology can be used to produce these hydrophobins in adequate amounts for commercial applications. Further, there is a need for rapid, simple methods of treating surfaces with hydrophobins. Accordingly, Applicants have isolated a DNA sequence encoding a novel hydrophobin from a thermophilic fungus, provided improved methods of surface treatment with the encoded hydrophobin protein, and expanded the variety of material surfaces for hydrophobin coating.